The present invention relates to a G protein-coupled receptor whose expression is regulated in hematopoietic cells upon activation and functions as a tumor suppressor gene.
The family of G protein-coupled receptors (GPCRs) has at least 250 members (Strader et al. FASEB J., 9:745-754, 1995; Strader et al. Annu. Rev. Biochem, 63:101-32, 1994). It has been estimated that one percent of human genes may encode GPCRs. GPCRs bind to a wide-variety of ligands ranging from photons, small biogenic amines (i.e., epinephrine and histamine), peptides (i.e., IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). Upon ligand binding, GPCRs regulate intracellular signaling pathways by activating guanine nucleotide-binding proteins (G proteins). GPCRs play important roles in diverse cellular processes including cell proliferation and differentiation, leukocyte migration in response to inflammation, and cellular response to light, odorants, neurotransmitters and hormones (Strader et al., supra.).
Interestingly, GPCRs have functional homologues in human cytomegalovirus and herpesvirus, suggesting that GPCRs may have been acquired during evolution for viral pathogenesis (Strader et al., FASEB J., 9:745-754, 1995; Arvanitakis et al. Nature, 385:347-350, 1997; Murphy, Annu. Rev. Immunol. 12:593-633, 1994).
The importance of G protein-coupled receptors is further highlighted by the recent discoveries that its family members, chemokine receptors CXCR4/Fusin and CCR5, are co-receptors for T cell-tropic and macrophage-tropic HIV virus strains respectively (Alkhatib et al., Science, 272:1955, 1996; Choe et al., Cell, 85:1135, 1996; Deng et al., Nature, 381:661, 1996; Doranz et al., Cell, 85:1149, 1996; Dragic et al., Nature, 381:667 (1996); Feng et al., Science 272:872, 1996). It is conceivable that blocking these receptors may prevent infection by the human immunodeficiency (HIV) virus.
BCR-ABL is a chimeric tyrosine kinase oncogene generated by a reciprocal chromosomal translocation t(9;22). This chimeric oncogene found in Ph1-positive stem cells is associated with the pathogenesis of chronic myelogenous leukemia and acute lymphocytic leukemia. Mutational analysis has defined critical domains within BCR-ABL important for its functions. In particular, inactivation of the SH2 domain greatly reduced the malignant and leukemogenesis potential of BCR-ABL in vivo.
One embodiment of the present invention is an isolated polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 or 3.
The present invention also provides an isolated polynucleotide encoding a G protein-coupled receptor (GPCR) regulated by a protein tyrosine kinase, wherein said polynucleotide is capable of hybridizing to a polynucleotide having the sequence shown in SEQ ID NO: 1 at 65xc2x0 C. in 2xc3x97SSC, 0.1% SDS.
Another embodiment of the invention is an isolated, recombinant protein tyrosine kinase-regulated GPCR having the amino acid sequence shown in SEQ ID NO: 2 or 4. Preferably, the protein having the amino acid sequence shown in the SEQ ID NO: 2 is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 1. Advantageously, the protein having the amino acid sequence shown in the SEQ ID NO: 4 is obtained by expression of a polynucleotide having the sequence shown in SEQ ID NO: 3.
The present invention also provides an isolated GPCR encoded by a polynucleotide having the sequence shown in SEQ ID NO: 1 at 65xc2x0 C. in 2xc3x97SSC, 0.1% SDS.
Another embodiment of the invention is isolated antibodies to the GPCR described above. Preferably, the antibodies are polyclonal. Alternatively, the antibodies are monoclonal.
Another embodiment of the invention is a method of identifying a compound which activates the protein tyrosine kinase-regulated GPCR, comprising the steps of contacting the GPCR with a test compound; determining whether the compound binds to the GPCR; and testing compounds which bind to said GPCR in a receptor activity assay, whereby stimulation of receptor activity indicates that the compound is an activator of the GPCR Preferably, the GPCR is expressed on the cell surface. Advantageously, the receptor activity assay is fibroblast transformation, bone marrow transformation, cell cycle analysis, in vivo tumor formation or in vivo leukemogenesis.
The present invention also provides a method of identifying a compound which inhibits the protein tyrosine kinase-regulated GPCR, comprising the steps of: contacting the GPCR with a test compound; determining whether the compound binds to the GPCR; and testing compounds which bind to the GPCR in a receptor activity assay, whereby inhibition of receptor activity indicates that the compound is an inhibitor of the GPCR. Preferably, the GPCR is expressed on the cell surface. Advantageously, the receptor activity assay is fibroblast transformation, bone marrow transformation, cell cycle analysis, in vivo tumor formation or in vivo leukemogenesis.
Another embodiment of the invention is a method of identifying a compound which activates the protein tyrosine kinase-regulated GPCR, comprising the steps of: contacting the GPCR with a test compound; determining whether the compound binds to the GPCR; and testing compounds which bind to the GPCR in a receptor activity assay, whereby activation of receptor activity indicates that the compound is an inhibitor of the GPCR. Preferably, the GPCR is expressed on the cell surface. Advantageously, the receptor activity assay is fibroblast transformation, bone marrow transformation, cell cycle analysis, in vivo tumor formation or in vivo leukemogenesis.